Lyophilized Cyclophosphamide Composition and Methods of Preparation Thereof

ABSTRACT

An improved lyophilized cyclophosphamide solid composition is described. The lyophilized cyclophosphamide solid composition is thermally stable, contains anhydrous cyclophosphamide and mannitol, and is substantially free of cyclophosphamide monohydrate. A method for preparing the lyophilized cyclophosphamide solid composition is also provided.

BACKGROUND OF THE INVENTION

Cyclophosphamide is a synthetic antineoplastic drug which is chemicallyrelated to the nitrogen mustards. Cyclophosphamide, having the chemicalname 2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine2-oxide, can exist in both monohydrate and anhydrous forms as shown inFormula I and Formula II below:

The monohydrate form is preferred for pharmaceutical processing, as theanhydrous form was reported to be highly unstable and readily picks upwater to form the monohydrate when exposed to a relative humidity ofabout 20-30% or higher at about 25° C. The monohydrate drug form isstable but, under dry conditions (relative humidity of about 20% orless), begins to lose the water of hydration, giving rise to unwanteddegradation products which may potentially impact product quality andend-use limitation. Accordingly, maintaining proper manufacturing andstorage conditions are essential to product quality attributes andcritical for application for approved regulatory usage.

Cyclophosphamide is available in sterile parenteral dosage formulationsconsisting of sterile packaged dry powder blend admixtures ofcyclophosphamide monohydrate and sodium chloride, which are dissolved inwater prior to administration. However, such formulations havedisadvantages, such as glassiness and/or stickiness acquired by thepowder premix composition during the processing and storage, which mightimpact overall product quality and end-use applicability, includinginferior solubility and decreased potency, as well as deteriorationduring storage.

Lyophilization, or freeze drying, is a process for creating or isolatinga stable preparation of a solid product by freezing and dehydrationand/or removal of solvents utilized for dissolution of the frozenproduct under vacuum. The sublimation of the ice from the frozensolution leaves the solid, dried components of the original liquid.

U.S. Pat. No. 4,537,883 to Alexander et al. discloses that a lyophilizedcyclophosphamide solid composition containing 4% moisture gave a productwith superior thermal stability, compared to lyophilizedcyclophosphamide solid compositions with moisture levels of 1% or less,or even cyclophosphamide itself. It describes two methods to prepare thesolid compositions. One method involves lyophilization of a sterilesolution of cyclophosphamide, mannitol and water in a short period oftime (24 hours) to produce a “dry” lyophilizate having a moisturecontent of 2% or less, then humidifying the “dry” lyophilizate to give a“hydrated” lyophilizate containing approximately 4% moisture. The othermethod involves lyophilization of the sterile solution using a longer 2stage lyophilization process (48 hours), where the first stage offreeze-drying was done with a low condenser temperature of −60° C. and ashelf temperature at 10° C., and when the product temperature (measuredby thermocouple) reached about −12° C., the second stage offreeze-drying was conducted by raising the condenser temperature to −30°C. and lowering the shelf temperature to −10° C.

U.S. Pat. No. 4,659,699 to Daniel et al. discloses a two-stage processinvolving freeze drying an aqueous solution of cyclophosphamide with anexcipient until the moisture content is less than 2% by weight, thenrehydrating the freeze dried material to the moisture content in thecritical range of 2-7% by weight.

U. S. Patent Application Publication No. 2015/0290226 of Chandrashekharet al. describes a lyophilization process that did not need rehydrationstep and produced crystalline monohydrate form containing 5.7 to 6.8% ofwater. The lyophilization was conducted with one or more organicsolvents or a mixture of organic solvent(s) and water.

Many known lyophilized preparations of cyclophosphamides or similardrugs contain components such as bulking or matrix forming agents. Theseadditives include salts such as sodium chloride, mono-, di- andpolysaccharides, and sugar alcohols such as mannitol. D-mannitol isknown to exist as three polymorphs: α (also known as κ), β, and δ.Preparations which are mannitol free are also known. For example, U.S.Pat. No. 5,036,060 of Alam et al. describes a mannitol-free lyophilizedformulation of cyclophosphamide. The formulation is prepared from asolution of cyclophosphamide, water, and sodium chloride, and involvesperforming several freezing and cooling steps, applying vacuum, anddrying until a target moisture content is achieved.

Many prior art processes require a rehydration step at the end ofprocessing, and others require the use of solvents other than water.These processes are disadvantageous for several reasons. For examples, arehydration step may result in significant variability in the moisturelevel from vial to vial within a single batch, as well as variabilityfrom batch to batch. The use of solvents other than water introducesadditional residual materials that must be quantified and controlled.

There is a need for improved lyophilized cyclophosphamide compositionsand improved methods for lyophilizing cyclophosphamide.

BRIEF SUMMARY OF THE INVENTION

It was unexpectedly discovered in the present invention that an improvedlyophilized cyclophosphamide solid composition containing anhydrouscyclophosphamide and mannitol is thermally stable over extended storageperiods, even when the composition has a moisture content less than 1%by weight. The solid composition contains one or more crystalline formsthat cannot be attributed to any of the known forms of cyclophosphamideor mannitol.

The solid cyclophosphamide composition can be obtained by an improvedlyophilization process that does not require a rehydration step or anorganic solvent. Different from the two stage lyophilization processdescribed in U.S. Pat. No. 4,537,883, the present lyophilization processdoes not require measuring the temperature of the product duringfreeze-drying, nor a second stage freeze drying at a condensertemperature of −30° C. and a shelf temperature of −10° C. Moreover, thelyophilized composition obtained from the present lyophilization processcontains a moisture level much less than 4% by weight as that obtainedfrom the lyophilization process of U.S. Pat. No. 4,537,883.

Accordingly, in one general aspect, the invention relates to alyophilized cyclophosphamide solid composition comprisingcyclophosphamide and mannitol, wherein the composition contains one ormore crystalline forms exhibiting an X-ray powder diffraction (XRPD)spectrum having one or more peaks at diffraction angle (2θ) of 8.1±0.2°,8.5±0.2°, 12.3±0.2° or 13.4±0.2°, preferably a peak at 20 of 8.5±0.2°.In one embodiment of the invention, the lyophilized cyclophosphamidesolid composition contains one or more crystalline forms exhibiting anXRPD spectrum having the peaks at diffraction angle (2θ) of 8.1±0.2°,8.5±0.2°, 12.3±0.2° and 13.4±0.2°. The assignments for thesecharacteristic peaks are determined by reference XRPD diffractograms.

In one embodiment of the invention, the lyophilized cyclophosphamidesolid composition has a moisture content of less than 2% by weight,preferably less than 1% by weight. Preferably, the composition consistsessentially of anhydrous cyclophosphamide and mannitol. More preferably,the lyophilized cyclophosphamide solid composition is substantially freeof cyclophosphamide monohydrate.

In another embodiment of the invention, the lyophilized cyclophosphamidesolid composition has a weight ratio of cyclophoshamide anhydrous tomannitol within a range of about 0.05 to 10, more preferably about 0.05to 5, even more preferably about 1.2 to 1.8, most preferably about 1.3(1 part mannitol to 1.33 parts anhydrous cyclophosphamide).

In a preferred embodiment of the invention, the lyophilizedcyclophosphamide solid composition consists essentially ofcyclophosphamide and mannitol, wherein the composition contains one ormore crystalline forms having an X-ray powder diffraction (XRPD)spectrum comprising a peak at 2θ of 8.5±0.2°, preferably peaks at 2θ of8.5±0.2°, 8.1±0.2°, 12.3±0.2° and 13.4±0.2°. The cyclophosphamide can bepredominantly or only present in the anhydrous form.

In another general aspect, the invention relates to a process forproducing a lyophilized cyclophosphamide solid composition. The processdoes not involve a rehydration step, and comprises:

-   -   (1) preparing a solution containing cyclophosphamide, mannitol,        and water;    -   (2) filling a receptacle with the solution;    -   (3) inserting the receptacle filled with the solution into a        lyophilizer having a first shelf temperature of 0° C. to 25° C.,        and maintaining the first shelf temperature to allow all the        solution to equilibrate at the first shelf temperature;    -   (4) cooling the lyophilizer to a second shelf temperature of        −75° C. to −30° C. at a first controlled rate, and maintaining        the second shelf temperature to allow the solution to form ice        and to allow the temperature of the ice to reach a steady state;    -   (5) chilling the condenser of the lyophilizer to a temperature        below −40° C., preferably −50° C. or below;    -   (6) evacuating the chamber of the lyophilizer to a pressure of        less than 4500 μm Hg;    -   (7) warming the lyophilizer to a third shelf temperature of        −30° C. to 40° C. at a second controlled rate, and maintaining        the third shelf temperature and the chamber pressure at the        pressure of less than 4500 μm Hg to allow sublimation of the ice        and thereby produce a sublimed composition; and    -   (8) setting the lyophilizer to a fourth shelf temperature of        0° C. to 40° C., and maintaining the fourth shelf temperature        and the chamber pressure at the pressure of less than 4500 μm Hg        to desorb remaining moisture and thereby produce the lyophilized        cyclophosphamide solid composition.

In one embodiment of the invention, a solution containingcyclophosphamide, mannitol and water, is prepared by a methodcomprising:

-   -   (a) adding mannitol to water to obtain a mannitol solution;    -   (b) chilling the mannitol solution to 0° C. to 20° C.; and    -   (c) admixing cyclophosphamide monohydrate to the mannitol        solution at 0° C. to 20° C. to obtain the solution containing        cyclophosphamide, mannitol, and water.

In one embodiment of the invention, the cyclophosphamide monohydrateused for the preparation of the lyophilization solution has a particlesize that facilitates the efficient dissolution of cyclophosphamidemonohydrate, preferably, the cyclophosphamide monohydrate has a d(0.5)particle size distribution of 300 μm to 800 μm, more preferably 100 to400 μm.

In another embodiment of the invention, the lyophilization solutioncontains 5 to 25 mg/mL cyclophosphamide monohydrate and 5 to 25 mg/mLmannitol

Preferably, the solution containing cyclophosphamide, mannitol and wateris sterilized, for example, by filter sterilization. More preferably,the lyophilization solution is prepared, sterilized, filled in thereceptacle, and placed in the lyophilizer under conditions withoutsignificant degradation of cyclophosphamide.

According to embodiments of the invention, the lyophilizer is precooledto a first shelf temperature before the cyclophosphamide solution isloaded. Preferably, the first shelf temperature is 0° C. to 25° C., suchas 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25° C. After the cyclophosphamide solution isloaded to the lyophilizer, the first shelf temperature is maintained,for example, for 1 to 15 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 hours or any time periods in between, to allow allthe solution to equilibrate at the first shelf temperature.

The lyophilizer is then chilled to a second shelf temperature of −75° C.to −30° C., such as −75, −74, −73, −72, −71, −70, −69, −68, −67, −66,−65, −64, −63, −62, −61, −60, −59, −58, −57, −56, −55, −54, −53, −52,−51, −50, −49, −48, −47, −46, −45, −44, −43, −42, −41, −40, −39, −38,−37, −36, −35, −34, −33, −32, −31, or −30° C., at a controlled rate,such as 6 to 120° C. per hours, more preferably at, but not limited to,25° C., 30° C. or 35° C. per hour. The second shelf temperature ismaintained for a period of time sufficient to allow the solution to formice and to allow the temperature of the ice to reach a steady stateprior to proceeding to evacuation. For example, the second shelftemperature can be maintained for 1-20 hours, such as 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours or any timeperiods in between.

The condenser of the lyophilizer is chilled to a temperature below −40°C., preferably −50° C. or below, such as −45, −50, −55, −60° C., or evenlower. The chamber of the lyophilizer is evacuated to a pressure of lessthan about 4500 μm Hg, such as pressures of 150 to 250 μm Hg. Thepressure is maintained by, for example, but not limited to, bleeding innitrogen gas that is filtered through a 0.1 or 0.2 μm filter.

For the primary drying, the lyophilizer is warmed to a third shelftemperature at a second controlled rate. The third shelf temperature isselected to drive sublimation while maintaining the product temperaturesin a safe range to prevent a change upon completion of sublimation. Thecontrolled rate is selected to prevent breakage of the container (e.g.,vials) during the ramp. Preferably, the third shelf temperature is −30°C. to 40° C., such as −30, −29, −28, . . . 39, or 40° C., morepreferably 5 to 15° C., and the second controlled rate is 6° C. to 120°C. per hour, such as, but not limited to, 10° C., 15° C. or 20° C. perhour. The chamber pressure is maintained under vacuum (e.g., at thepressure of less than about 4500 μm Hg) for a period of time sufficientfor all the product temperatures to raise and reach a steady state at ornear the third shelf temperature, e.g., to allow sublimation of the iceto thereby produce a sublimed composition. For example, the chamberpressure can be maintained under vacuum for 10-360 hours.

The sublimed composition is further dried at a fourth shelf temperatureof 0° C. to 40° C., such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40° C., under vacuum. Thisadditional drying step desorbs remaining moisture to thereby produce alyophilized cyclophosphamide solid composition of the invention. Theadditional drying step can be conducted for, e.g., 3 to 12 hours, or alonger period of time as needed.

The lyophilization process is stopped by raising the pressure of thechamber of the lyophilizer, for example, to about 1 to about 14.7 psia,more preferably about 10.0 psia, and stoppering the receptaclecontaining the lyophilized cyclophosphamide solid composition.

Another aspect of the present invention relates to a lyophilizedcyclophosphamide preparation that is prepared by a method of theinvention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. These drawings are included for the purpose ofillustrating a preferred embodiment of the invention. The invention isnot limited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 is an XRD spectrum of a lyophilized cyclophosphamide compositionaccording to an embodiment of the invention; and

FIG. 2 is an XRPD pattern comparison of lyophilized cyclophosphamidecompositions according to embodiments of the invention with known formsof cyclophosphamide and mannitol; the analyzed samples are, from top tobottom:

-   -   1. a lyophilized cyclophosphamide composition according to a        first embodiment of the invention;    -   2. a lyophilized cyclophosphamide composition according to a        second embodiment of the invention;    -   3. a lyophilized cyclophosphamide composition according to a        third embodiment of the invention;    -   4. a lyophilized cyclophosphamide composition according to a        fourth embodiment of the invention;    -   5. cyclophosphamide anhydrous racemate form (Cyclophosphamide        racemate anhydrate, Jones, et al., Acta Crystallogr., Sect. C,        vol. 52, p 2359, 1996);    -   6. S-cyclophosphamide P1 form (Adamiak et al., J. Biosci., 32,        672, 1977);    -   7. cyclophosphamide anhydrous enantiopure form (Cyclophosphamide        enantiopure: Karle et al., J. Am. Chem. Soc., vol. 99, p 4803,        1977);    -   8. delta D-mannitol (Mannitol delta: Botez et al, Powder Diffr.,        vol. 18, p 214, 2003);    -   9. kappa D-mannitol (The Crystal Structure of K Form of        D-Mannitol, Kim et al., Acta. Cryst. B24, 1449-1455, 1968);    -   10. mannitol hemihydrate (Mannitol hemihydrate, Nunes et al., J.        Pharm. Sci., vol. 93, p 2800, 2004);    -   11. beta D-mannitol (Mannitol beta, Kaminsky et al.,        Kristallogr., vol. 212, p 283, 1997); and    -   12. cyclophosphamide monohydrate (cyclophosphamide racemate        monohydrate, Clardy et al., Phosphorus, vol. 4, p 151, 1974).

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the specification is for the purpose of providing contextfor the invention. Such discussion is not an admission that any or allof these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification. All patents,published patent applications and publications cited herein areincorporated by reference as if set forth fully herein. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the invention.

Unless otherwise stated, any numerical value, such as a concentration, atemperature, a period of time, or a range described herein, are to beunderstood as being modified in all instances by the term “about.” Thus,a numerical value typically includes ±10% of the recited value. Forexample, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v)to 11% (w/v). As used herein, the use of a numerical range expresslyincludes all possible subranges, all individual numerical values withinthat range, including integers within such ranges and fractions of thevalues unless the context clearly indicates otherwise.

Lyophilized Cyclophosphamide Composition

It was previously believed that the monohydrate form of cyclophosphamidewas necessary for a stable product, but this assumption has beendisproven in the present invention. It is surprisingly discovered in thepresent invention that a lyophilized cyclophosphamide solid compositioncontaining anhydrous cyclophosphamide and mannitol is thermally stableover extended storage periods. The composition has a moisture contentless than 2% by weight, preferably less than 1% by weight. Thecomposition contains one or more crystalline forms that cannot beattributed to any of the known forms of cyclophosphamide or mannitol.

According to one embodiment of the invention, a lyophilizedcyclophosphamide solid composition contains a mixture ofcyclophosphamide anhydrous and mannitol polymorphs, including one ormore novel crystalline forms exhibiting an X-ray powder diffraction(XRPD) spectrum having one or more peaks at diffraction angle (2θ) of8.1±0.2°, 8.5±0.2°, 12.3±0.2° or 13.4±0.2°, preferably a 20 of 8.5±0.2°.In one embodiment, the lyophilized cyclophosphamide solid compositioncontains one or more novel crystalline forms having a XRPD spectrumcomprising peaks at 2θ of 8.1±0.2°, 8.5±0.2°, 12.3±0.2° and 13.4±0.2°.In a preferred embodiment of the invention, the lyophilized compositionis substantially free of cyclophosphamide monohydrate.

More specifically, XRPD (X-ray Powder Diffraction), DVS (Dynamic VaporSorption) analysis, and HSM (hot stage microscopy) were performed toevaluate the crystallinity of compositions according to embodiments ofthe invention, and determine the components present in the compositions.It was found that compositions according to embodiments of the inventioncontain anhydrous cyclophosphamide and mannitol and more specificallycontain anhydrous cyclophosphamide in racemate form and δ D-mannitol.Compositions according to embodiments of the present invention canfurther contain κ D-mannitol, β D-mannitol hemihydrate, and/or anhydrouscyclophosphamide in enantiopure form, depending on the particularsample. Surprisingly, the inventive compositions contain one or morecrystalline forms that cannot be attributed to any of the known forms ofcyclophosphamide or mannitol. The one or more novel crystalline formsexhibit an XRPD spectrum having one or more peaks at 2θ of 8.1±0.2°,8.5±0.2°, 12.3±0.2° or 13.4±0.2°. Preferably, an inventive compositionaccording to an embodiment of the present invention is substantiallyfree of cyclophosphamide monohydrate, and no characteristic peakscorresponding to cyclophosphamide monohydrate is detected on the XRPDspectrum of the composition. The weight ratio of total cyclophosphamideto total mannitol in the composition is preferably 0.5 to 10, even morepreferably 1.2 to 1.8, more preferably about 1.3.

The characteristic peaks corresponding to components of a compositionaccording to an embodiment of the invention can be seen in therepresentative XRD spectrum in FIG. 1. As shown in FIG. 2, no peak whichcorresponds to cyclophosphamide monohydrate is detected in thecompositions according to embodiments of the present invention.

When a composition contains cyclophosphamide monohydrate, if the watercontent is not correctly controlled, there is a potential for excesswater that would begin to degrade the cyclophosphamide during storage.Accordingly, inventive lyophilized cyclophosphamide solid compositionsaccording to embodiments of the invention, which are substantially freeof water or cyclophosphamide monohydrate, are advantageous because thereis no potential for this degradation during storage. The inventivecompositions have been shown to exhibit long term stability for longerthan 3 months when stored at 5° C. or at 25° C./60% RH (relativehumidity).

Lyophilization Process

The solid cyclophosphamide composition of the invention can be obtainedby an inventive lyophilization process. Different from the prior art,the lyophilization processes according to embodiments of the inventiondo not require a rehydration step or an organic solvent. It comprises:

-   -   (1) preparing a solution containing cyclophosphamide, mannitol,        and water;    -   (2) filling a receptacle with the solution;    -   (3) inserting the receptacle filled with the solution into a        lyophilizer having a first shelf temperature of 0° C. to 25° C.,        preferably 3° C. to 8° C., and maintaining the first shelf        temperature to allow all the solution to equilibrate at the        first shelf temperature;    -   (4) cooling the lyophilizer to a second shelf temperature of −75        to −30° C., preferably −55° C. to −40° C., at a first controlled        rate, and maintaining the second shelf temperature to allow the        solution to form ice and to allow the temperature of the ice to        reach a steady state;    -   (5) chilling condenser of the lyophilizer to a temperature below        −40° C., preferably −50° C. or below;    -   (6) evacuating chamber of the lyophilizer to a pressure of less        than 4500 μm Hg, preferably 150-250 μm Hg, and controlling the        chamber pressure at the pressure of less than 4500 μm Hg,        preferably 150-250 μm Hg, by bleeding in an inert gas;    -   (7) warming the lyophilizer to a third shelf temperature of −30        to 40° C., preferably 5° C. to 15° C., at a second controlled        rate, and maintaining third shelf temperature and the chamber        pressure at the pressure of less than 4500 μm Hg, preferably        150-250 μm Hg, to allow sublimation of the ice to thereby        produce a sublimed composition; and    -   (8) setting the lyophilizer to a fourth shelf temperature of        0° C. to 40° C., preferably 15° C. to 25° C., and maintaining        the fourth shelf temperature and the chamber pressure at the        pressure of less than 4500 μm Hg to desorb remaining moisture to        thereby produce the lyophilized cyclophosphamide solid        composition.

Solution Preparation

According to an embodiment of the invention, a solution consistingessentially of cyclophosphamide monohydrate, mannitol, and water isprepared. That is, no additional solvents are contained in the solution.The solution can be prepared by known methods with cyclophosphamideanhydrous or cyclophosphamide monohydrate. Different form of mannitol(powder or solution) can be employed.

It has been found in the invention that significant degradation ofcyclophosphamide can occur when the bulk solution temperature ismaintained at 5° C. to 15° C., such as 12° C.±3° C., for an extendedperiod of time. Therefore, it is preferable to achieve rapid dissolutionof cyclophosphamide to leave sufficient time for filtrating, filling,and loading of the solution into the lyophilizer before the significantdegradation occurs.

In a preferred embodiment of the invention, a rapid dissolution isachieved when mannitol powder is first added to water to form an aqueoussolution, followed by chilling the solution to 5° C. to 15° C., morepreferably 12° C.±3° C., and then adding the cyclophosphamidemonohydrate (USP grade) to the chilled mannitol solution. Theconcentration of mannitol in the solution is preferably 14 mg/ml to 19.6mg/ml. The concentration of cyclophosphamide monohydrate in the solutionis preferably 20 to 28 mg/mL, and is more preferably kept at or below 25mg/mL (equivalent to 23.4 mg/mL cyclophosphamide anhydrous). Suchconcentrations have been found to eliminate or minimize precipitation ofthe product from the solution, allowing for a clear, homogeneoussolution when both solutes have been added.

The cyclophosphamide solution contains cyclophosphamide (anhydrous) andmannitol in specified ratios and amounts. For example, the solution cancontain cyclophosphamide anhydrous and mannitol at a weight ratio of 0.5to 10, more preferably 0.5 to 5, even more preferably 1.2 to 1.5, mostpreferably 1.3. Such concentrations can be employed to produce vialshaving different strengths. For example, a vial containing 500 mgcyclophosphamide (534.5 mg cyclophosphamide monohydrate) contains 375 mgmannitol, a vial containing 1 g cyclophosphamide (1069.0 mgcyclophosphamide monohydrate) contains 750 mg mannitol, and a vialcontaining 2 g cyclophosphamide (2138.0 mg cyclophosphamide monohydrate)contains 1500 mg mannitol.

In a preferred embodiment of the invention, a rapid dissolution isachieved by using cyclophosphamide monohydrate having a preferredparticle size. The particle size can be characterized by the mediandiameter of particles of cyclophosphamide monohydrate used to preparethe solution. As used herein, the “d(0.5)” particle size distribution isthe median diameter of the particle size distribution, and representsthe particle size at which 50% of the particles are larger and 50% ofthe particles are smaller than the d(0.5) value. Similarly, the “d(0.1)”value is the particle size at which 10% of the particles are smaller and90% of the particles are larger, and the “d(0.9)” value is the particlesize at which 90% of the particles are smaller and 10% of the particlesare larger. The particle size can be measured using methods known in theart, for example, as determined in dry-dispersion mode using a MalvernMastersizer 2000 equipped with a Scirocco 2000 module (measuring range0.02 to 2000 μm).

Preferably, the cyclophosphamide monohydrate used for the preparation ofthe solution for lyophilization has a d(0.5) particle size distributionof 300-800 μm, preferably 100-400 μm, for the preparation of thesolution for lyophilization. The particle size of the cyclophosphamidemonohydrate can be further characterized as having a d(0.1) particlesize distribution of 40-180 μm, and/or as having a d(0.9) particle sizedistribution of about 300-800 μm.

In another preferred embodiment of the invention, a rapid dissolution isachieved by rapid mixing with a strong vortex, such as with a stir barand/or overhead stirrer. The temperature is preferably maintained at 5°C. to 15° C., more preferably at 12° C.±3° C., during the stirring.However, as noted above, other methods of preparing the cyclophosphamidesolution are also within the scope of the invention.

After the solution of mannitol and cyclophosphamide has been mixed, itis preferably filtered. In an exemplary embodiment, filtration isperformed through a 0.2 μm sterilizing filter with a polyvinylidenefluoride (PVDF) membrane, such as a Millipak 20 filter. Filtration isperformed to sterilize the bulk solution prior to performing an asepticfilling operation. This method of filtration is exemplary, not limiting,and other filtering methods and procedures which are known in the artwould also be equally applicable for the method of the invention. Thesolution is kept chilled at 5° C. to 15° C., more preferably at 12°C.±3° C., in the starting and receiving vessels during filtration.

The components of the solution, cyclophosphamide monohydrate andmannitol which meet USP requirements, are commercially available.Cyclophosphamide monohydrate is preferably stored at −2° C. to −8° C.prior to use.

Receptacle Preparation

According to an embodiment of the invention, the lyophilization processinvolves filling the desired receptacle, container or vessel with acyclophosphamide solution. The filling is performed at 5° C. to 15° C.,such as at 12° C.±3° C.

Different receptacles can be utilized, such as molded vials or tubing.For the purposes of this description, the terms “container,” “vial,” and“receptacle” will be used interchangeably. A variety of vial sizes canbe utilized, including, without limitation, 20 mL, 50 mL, and 100 mLmolded vials.

Each vial is filled with cyclophosphamide to a desired target quantityof cyclophosphamide depending on the concentration in the solution, suchas 10 mL for a 20 mL vial, 20 mL for a 50 mL vial, or 80 mL for a 100 mLvial. Methods of filling the vials or receptacles are known in the artand can be used in the invention in view of the present disclosure.

Chamber Filling and Pressurization

According to an embodiment of the invention, the lyophilization processinvolves inserting the vial(s) into a lyophilizer chamber having shelvesat a shelf temperature of 0° C. to 25° C., more preferably 3° C. to 8°C., even more preferably 5° C., for preventing nucleation (onset offreezing) and maintaining stability of cyclophosphamide in solution. Anylyophilizer can be utilized for the process of the invention, such as aHull Model 8FS15C pilot size lyophilizer. The vial(s) are placed ontothe shelves of the lyophilizer.

The chamber can be evacuated to greater than 10 psia, such as 12 psia,to ensure that the chamber is airtight.

Temperature Control

According to an embodiment of the invention, the lyophilization processinvolves maintaining the lyophilizer at the shelf temperature of 0° C.to 25° C., such as 3° C. to 8° C., or 5° C., and the pressure in thechamber at greater than 10 psia to equilibrate the temperature of thecyclophosphamide solution contained in the vials. The temperature of theshelves is controlled at a target set point of 0° C. to 25° C. until allof the product has been loaded onto the shelves. This temperature isthen held for an appropriate time so that all of the product samples canequilibrate to the target temperature. Such a time period can bedetermined by routine experimentation in view of the present disclosure.For example, it can be 1 to 15 hours. It is desirable to equilibrate theproduct to the target temperature so that all samples (receptacles) willhave the same thermal history prior to freezing.

Temperature Reduction and Maintenance

According to an embodiment of the invention, the lyophilizer shelves arechilled to a shelf temperature of −75° C. to −30° C. at an averagecontrolled rate, such as 6° C. to 120° C. per hour, for example, but notlimited to, 15, 20, 25 or 30° C. per hour, to ensure adequatesolidification of the product, for example, by keeping the product belowa minimum freezing temperature, such as −32° C. It has been found thatslow cooling of the cyclophosphamide solution, and a moderate ramp speedleads to a more uniform freezing throughout the vial by ensuring a moreuniform temperature distribution throughout the vial. In a preferredembodiment, the shelves are chilled at an average controlled rate of 30°C. per hour. Thus, the time required to cool the lyophilizer from theinitial shelf temperature of 0° C. to 25° C. to the second shelftemperature of −75° C. to −30° C. can be one to four hours.

After cooling, the temperature of the lyophilizer shelves are maintainedat −75° C. to −30° C. and the chamber is maintained at the pressure ofgreater than 10 psia, preferably at 11 to 13 psia, for additional timeto equilibrate the temperature of the cyclophosphamide solutioncontained in the receptacles and produce frozen solutions (ice). Theappropriate time required for equilibration and solidification can bedetermined by routine experimentation. For example, it can be 1 to 20hours. The time is selected to ensure that there is sufficient time forthe product temperature to reach a steady state prior to sublimation.

In one embodiment of the invention, an additional warming/coolingsequence for annealing is optionally performed after cooling to theshelf temperature of −75° C. to −30° C. and prior to chilling of thecondenser. Specifically, the shelves are warmed to a shelf temperatureof −30° C. to less than 0° C., preferably −20° C. to −10° C., at acontrolled rate, such as an average rate of 15° C. per hour or 30° C.per hour, and then cooled back to the shelf temperature of −75° C. to−30° C. at a controlled rate, such as an average rate of 15° C. per houror 30° C. per hour. This shelf temperature of −75° C. to −30° C. is thenmaintained to equilibrate the temperature of the cyclophosphamidesolution and produce a frozen solution, such as for 1 to 15 hours,preferably 3 to 4 hours.

Chilling of the Condenser and Evacuating the Chamber

The condenser of the lyophilizer is cooled to below −40° C., such as−50° C. or below, to ensure that enough water vapor can be stripped outof the air in the chamber to prevent fouling of the vacuum pump. Thelyophilizer chamber is then evacuated to a pressure of less than 4500microns of mercury (μm Hg), preferably 150-250 μm. The chamber pressureis then controlled in this range by, such as, but not limited to,bleeding in an inert gas such as the preferred nitrogen. Morepreferably, 0.1 or 0.2 micron filtered nitrogen NF is utilized tocontrol the chamber pressure at the desired level.

Sublimation

According to an embodiment of the invention, the ice in the frozencyclophosphamide solution is sublimed preferably by the following steps.

The sublimation process involves controlled gradual warming of thelyophilization shelves for the sublimation of the cyclophosphamidesolution. In a primary drying sequence, the shelves are warmed to −30°C. to 40° C., preferably 5° C. to 15° C. at a controlled rate, such asan average rate of 6° C. to 120° C. per hour, including, for example butnot limited to 10, 15 or 20° C. per hour, and then maintained at −30° C.to 40° C., preferably 5° C. to 15° C. for a sufficient time, such as30-190 hours, until all the ice has sublimed.

It has been found that holding the sample at a low temperature undervacuum for an extended time following sublimation results in apredominantly anhydrous compound. Thus, in a secondary drying sequence,the shelves are set to 0° C. to 40° C., preferably 0° C. to 10° C. or15° C. to 25° C., at a controlled rate such as 30° C. per hour orslower, and maintained at this temperature for, e.g., 3 to 12 hours, todesorb remaining moisture. The time needed for the final step can bedetermined by routine experimentation in view of the present disclosure.Secondary drying thus allows remaining moisture to be removed from theproduct by desorption while maintaining the product at a temperaturewell below the melting temperature observed by DSC (differentialscanning calorimetry).

Following sublimation, the resulting lyophilized cyclophosphamideproduct is a uniform, dense, white cake which, when reconstituted foruse, forms a clear, colorless solution.

The process according to the invention is a robust process which resultsin a lyophilized product that is thermally stable and can beconsistently manufactured. In contrast with lyophilized cyclophosphamidepreparations produced by known processes, the product according to theinvention includes one or more novel crystalline forms exhibiting anX-ray powder diffraction (XRPD) spectrum having one or more peaks atdiffraction angle (2θ) of 8.1±0.2°, 8.5±0.2°, 12.3±0.2° or 13.4±0.2°. Ithas a moisture level less than 2% by weight, preferably 1% by weight orless, and yet is still thermally stable. In a preferred embodiment ofthe invention, the lyophilized composition contains only the anhydrousform of cyclophosphamide with no evidence of the crystalline monohydrateform.

The invention will now be described in connection with the following nonlimiting examples.

Examples General Procedures

Cyclophosphamide monohydrate was obtained from AMPAC Fine Chemicals(Rancho Cordova, Calif.) and stored at 2° C. to 8° C. until use.Mannitol, USP, EP was obtained from Millipore, vials and tubing wereobtained from SGD or Thuringer, and stoppers were obtained from West. AHull Model 8FS15C pilot size lyophilizer was utilized for processing.

Cyclophosphamide solutions were formulated to contain mannitol,cyclophosphamide and water, and were filtered through a 0.2 μmsterilizing filter with a polyvinylidene fluoride membrane (Millipak 20filter), then filled into washed vials (volume 20 to 100 ml) to a targetfill quantity ranging from 200 mg/vial to 2000 mg/vial cyclophosphamideanhydrous. After loading the product, the lyophilizer was evacuated to12 psia and the desired lyophilization cycle was performed. Data wererecorded electronically every five minutes. Monitoring was performed inprocess using a Hastings Gauge (thermocouple vacuum gauge), whichindicates the end of sublimation in primary drying or the end ofdesorption in secondary drying. Following lyophilization, products werephysically inspected to evaluate the uniformity of appearance in color,texture, shape, and structure to assess the relative effects ofprocessing. Solutions were reconstituted using purified water andevaluated to determine the presence or absence of insoluble material,clarity, color, and time required to fully dissolve thecyclophosphamide. Moisture testing was performed using coulometric KarlFischer Titration, glass transition and/or melting point of solidmaterials was determined from high temperature differential scanningcalorimetry (HT-DSC), and residual moisture determination was performedusing thermogravimetric analysis (TGA).

Example 1: Investigation of Cyclophosphamide Dissolution

A series of experiments were performed in order to evaluate the effectsof order of addition, mixing method and speed, and impact ofcyclophosphamide monohydrate particle size distribution on thedissolution of cyclophosphamide. Various compounding methods wereinvestigated in order to achieve rapid dissolution within one hour inorder to leave sufficient time for filtration, filtering, and loading ofthe lyophilizer within twelve hours. The data obtained from thesestudies are found in Tables 1-3 below:

TABLE 1 Bulk Solution Concentrations Cyclo- Complete phosphamide Cyclo-Dissolution Monohydrate phosphamide Mannitol, USP, within time Study(mg/mL) (mg/mL) EP (mg/mL) limit? (yes/no) A 21.4 20.0 15.0 yes B 29.928.0 21.0 no C 29.9 28.0 21.0 no D 26.7 25.0 19.0 no E 29.9 28.0 21.0 noF 21.4 20.0 15.0 no G 29.9 28.0 21.0 yes H 28.0 26.2 21.0 no I 25.0 23.418.8 no J 25.0 23.4 18.8 no K 28.0 26.2 21.0 no L 25.0 23.4 18.8 yes M25.0 23.4 17.63 yes

TABLE 2 Order of Addition Order of Addition (Concentration) Rx 1^(st)2^(nd) 3^(rd) 1 Mannitol Chill Cyclophosphamide 2 Chill CyclophosphamideMannitol 3 Chill Cyclophosphamide Mannitol, added as a solution 4Mannitol Cyclophosphamide Chill

TABLE 3 Bulk Solution Properties Cyclophosphamide Anhydrous Order ofConcentration Dissolution Addition Study (mg/mL) (minutes) (see Table 2)Mixing Method A 20.0 105 Rx1 Stir Bar B 28.0 165 Rx1 Stir Bar C 28.0 54Rx1 Stir Bar D 28.0 59 Rx2 Stir Bar E 28.0 160 Rx3 Stir Bar F 28.0 21Rx4 Stir Bar G 28.0 190 Rx1 Stir Bar H 25.0 69 Rx1 Stir Bar I 28.0 267Rx1 Stir Bar and overhead J 20.0 63 Rx1 Stir Bar K 28.0 98 Rx2 Stir BarL 26.2 170 Rx1 Stir Bar M 26.2 111 Rx1 Overhead N 26.2 151 Rx1 OverheadO 23.4 60 Rx1 Overhead P 23.4 108 Rx2 Overhead Q 26.2 129 Rx2 Overhead R23.4 90 Rx1 Overhead S 23.4 35 Rx1 Overhead T 23.4 40 Rx1 Overhead

From these studies, it was determined that the preferred dissolutionmethod (Rx1 in Table 2) involves first adding mannitol powder to waterto form an aqueous solution, followed by chilling the solution and thenadding the cyclophosphamide monohydrate to the chilled mannitolsolution. The concentration of cyclophosphamide monohydrate ispreferably 25.0 mg/ml or lower, and particle size is also preferablycontrolled. Table 3b lists the particle size distributions of somebatches of cyclophosphamide monohydrate used in the preparation of thesolution, wherein the particle size distribution was measured using aMalvern particle size analyzer. In addition, rapid mixing with a strongvortex is also preferred to achieve rapid dissolution.

TABLE 3b Particle size distribution of cyclophosphamide monohydrateParticle Size Distribution (μm) Batch No. d (0.1) d (0.5) d (0.9) 1 75261 653 2 166 317 547 3 175 362 667 4 65 256 781 5 48 131 331

Example 2: Investigation of Filling and Lyophilization ofCyclophosphamide: 2000 mg Presentations

2000 mg/vial presentations of cyclophosphamide were prepared having,e.g., 25 mg/ml cyclophosphamide monohydrate (equivalent to 23.5 mg/mlcyclophosphamide), 17.6 mg/ml mannitol, and water. A bulkcyclophosphamide solution was prepared at a temperature 10° C. with adissolution time of about 68 minutes. The solutions were filled intovials, loaded onto the shelves of a lyophilizer at 5° C., and thechamber was evacuated to within 11-13 psia after loading. Tables 4a, 4b,5a and 5b list freeze drying parameters of exemplary processes forlyophilization of 2000 mg/vial presentations.

TABLE 4a Summary of Target Lyophilization Cycle Parameters: (2000 mg)Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours) (°C./hour) Pressure Setpoint Product Loading 5 4.0 Evac. to 12 PSIA to30.0 ensure chamber Freezing −50 6.0 is airtight 15.0 Primary Drying 10182.0 200 microns of Hg 30.0 Secondary Drying 20 10.0 Stoppering 20 —10.0 PSIA

TABLE 4b Summary of Target Lyophilization Cycle Parameters: (2000 mg)Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours) (°C./hour) Pressure Setpoint Product Loading 6 4.0 Evac. To 12 PSIA 30.0to ensure chamber Freezing −52 6.0 is airtight 15.0 Primary Drying 10175.0 200 microns of Hg 30.0 Secondary Drying 5 17 Stoppering 5 — 10.0PSIA

TABLE 5a Summary of Target Lyophilization Cycle Parameters: (2000 mg)Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours) (°C./hour) Pressure Setpoint Product Loading 5 4.0 Evac. To 12 PSIA 30.0to ensure chamber Freezing −50 6.0 is airtight 15.0 Primary Drying 12160.0 200 microns of Hg 30.0 Secondary Drying 20 4.0 Stoppering 20 —10.0 PSIA

Physical appearance for the product vials showed a uniform, dense, whitecake with no side shrinkage. Reconstitution was performed with PurifiedWater, USP. The reconstituted samples resulted in clear, colorlesssolutions. The pH dropped from 4.8 to 3.5-3.6 post-lyophilization.

Example 3: Investigation of Filling and Lyophilization ofCyclophosphamide: 500 mg Presentations

500 mg/vial presentations of cyclophosphamide were prepared having,e.g., 25 mg/ml cyclophosphamide monohydrate (equivalent to 23.4 mg/mlcyclophosphamide), 17.5 mg/ml mannitol, and water. A bulkcyclophosphamide solution was prepared at a temperature 10-11° C. with adissolution time of about 45 minutes. The bulk solutions were filled andloaded onto the shelves of a lyophilizer at 5° C., and the chamber wasevacuated to within 11-13 psia after loading. Tables 6a, 6b, 7a and 7blist freeze drying parameters of exemplary processes for lyophilizationof 500 mg/vial presentations.

TABLE 6a Summary of Target Lyophilization Cycle Parameters: (500 mg)Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours) (°C./hour) Pressure Setpoint Product Loading 5 3.0 Evac. To 12 PSIA 30.0to ensure chamber Freezing −50 4.0 is airtight 15.0 Primary Drying 1050.0 200 microns of Hg 30.0 Secondary Drying 20 8.5 Stoppering 20 — 10.0PSIA

TABLE 6b Summary of Target Lyophilization Cycle Parameters: (500 mg)Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours) (°C./hour) Pressure Setpoint Product Loading 5 3.0 Evac. To 12 PSIA 30.0to ensure chamber Freezing −50 4.0 is airtight 15.0 Primary Drying 1240.0 200 microns of Hg 30.0 Secondary Drying 5 10 Stoppering 5 — 10.0PSIA

Physical appearance for the product vials showed a uniform, dense, whitecake with no side shrinkage. Reconstitution was performed with PurifiedWater, USP. The reconstituted samples resulted in clear, colorlesssolutions. The pH dropped from 4.1 to 3.9 post-lyophilization. KarlFisher results showed an average moisture level of less than 1% (w/w),such as 0.34% (w/w).

Example 4: Investigation of Filling and Lyophilization ofCyclophosphamide: 200 mg Presentations

200 mg/vial presentations of cyclophosphamide was prepared having, e.g.,25 mg/ml cyclophosphamide monohydrate (equivalent to 23.4 mg/mlcyclophosphamide), 17.5 mg/ml mannitol, and water. A bulkcyclophosphamide solution was prepared at a temperature 10-14° C. with adissolution time of about 38 minutes. The bulk solutions were filledinto vials, loaded onto the shelves of a lyophilizer at 5° C., and thechamber was evacuated to within 11-13 psia after loading. Tables 8a, 8b,9a and 9b list freeze drying parameters of exemplary processes forlyophilization of 200 mg/vial presentations.

TABLE 8a Summary of Target Lyophilization Cycle Parameters: (200 mg)Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours) (°C./hour) Pressure Setpoint Product Loading 5 2.5 Evac. To 12 PSIA 30.0to ensure chamber Freezing −50 3.0 is airtight 15.0 Primary Drying 1032.0 200 microns of Hg 30.0 Secondary Drying 20 4.0 Stoppering 20 — 10.0PSIA

TABLE 8b Summary of Target Lyophilization Cycle Parameters: (200 mg)Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours) (°C./hour) Pressure Setpoint Product Loading 5 2.5 Evac. To 12 PSIA 30.0toensure chamber Freezing −50 3.0 is airtight 15.0 Primary Drying 1227.0 200 microns of Hg 30.0 Secondary Drying 5 2 Stoppering 5 — 10.0PSIA

TABLE 9a Summary of Target Lyophilization Cycle Parameters: (200 mg)Shelf Temp. Soak Ramping Setpoint Time Rate Step (° C.) (hours) (°C./hour) Pressure Setpoint Product Loading 6 3 Evac. To 12 PSIA 30.0 toensure chamber Freezing −55 2.0 is airtight 15.0 Primary Drying 8 32.0200 microns of Hg 30.0 Secondary Drying 5 4.0 Stoppering 5 — 10.0 PSIA

Physical appearance for the product vials showed a uniform, dense, whitecake with no side shrinkage. Reconstitution was performed with PurifiedWater, USP. The reconstituted samples resulted in clear, colorlesssolutions. The pH dropped from 4.8 to 3.9-4.0 post-lyophilization. Theaverage residual moisture is less than 1% (w/w), such as about 0.36%(w/w).

Example 5: Physical Characterization of Lyophilized CyclophosphamideCompositions

Several samples of lyophilized cyclophosphamide were analyzed using XRPD(X-ray Powder Diffraction), DVS (Dynamic Vapor Sorption) analysis, andHSM (hot stage microscopy) to evaluate the crystallinity and determinethe components present in the samples. An XRPD spectrum from onerepresentative sample is shown in FIG. 1.

FIG. 2 shows the XRPD pattern comparison of four lyophilizedcyclophosphamide compositions according to embodiments of the invention(the top four spectra) with known forms of non-GMP cyclophosphamide andmannitol. It was determined that the lyophilized cyclophosphamidecompositions according to embodiments of the invention all contain anunidentified peak at 8.5° (2θ), and additionally one or more peaks at8.1°, 12.3° and 13.4° (2θ), representing one or more novel crystal formsthat cannot be attributed to any of the known forms of cyclophosphamideor mannitol. The compositions contained anhydrous cyclophosphamide andmannitol, more specifically cyclophosphamide anhydrous enantiopure formand δ D-mannitol. Additionally, depending on the particular processsteps used for preparation, the compositions further contained κD-mannitol, β D-mannitol hemihydrate, and/or cyclophosphamide anhydrousracemate form in varying ratios. However, the compositions weresubstantially free of cyclophosphamide monohydrate.

It will be appreciated by those skilled in the art that changes could bemade to the embodiment described above without departing from the broadinventive concepts thereof. Also, based on this disclosure, a person ofordinary skill in the art would further recognize that the relativeproportions of the components illustrated could be varied withoutdeparting from the spirit and scope of the invention. It is understood,therefore, that this invention is not limited to the particularembodiment disclosed, but it is intended to cover modifications withinthe spirit and scope of the present invention as defined by the appendedclaims.

1. A lyophilized cyclophosphamide solid composition comprisingcyclophosphamide and mannitol, wherein the composition contains one ormore crystalline forms exhibiting an X-ray powder diffraction (XRPD)spectrum having a peak at diffraction angle (2θ) of 8.5±0.2, and thecomposition has a moisture content of less than 2% by weight.
 2. Thelyophilized cyclophosphamide solid composition of claim 1 having amoisture content of less than 1% by weight.
 3. The lyophilizedcyclophosphamide solid composition of claim 1, being substantially freeof cyclophosphamide monohydrate.
 4. The lyophilized cyclophosphamidesolid composition of claim 1, containing cyclophosphamide anhydrousenantiopure form and 6 D-mannitol.
 5. A lyophilized cyclophosphamidesolid composition consisting essentially of cyclophosphamide andmannitol, wherein the composition contains one or more crystalline formshaving an X-ray powder diffraction (XRPD) spectrum comprising a peak atdiffraction angle (2θ) of 8.5±0.2°, and the composition has a moisturecontent of less than 2% by weight.
 6. The lyophilized cyclophosphamidesolid composition of claim 5, being substantially free ofcyclophosphamide monohydrate.
 7. The lyophilized cyclophosphamide solidcomposition of claim 6, having a weight ratio of mannitol tocyclophoshamide anhydrous of 1.2 to 1.8. 8.-24. (canceled)
 25. Thelyophilized cyclophosphamide solid composition of claim 1, wherein theXRPD spectrum further comprises peaks at diffraction angle (2θ) of8.1±0.2°, 12.3±0.2°, and 13.4±0.2°.
 26. The lyophilized cyclophosphamidesolid composition of claim 5 having a moisture content of less than 1%by weight.